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1. Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems

   https://doi.org/10.1038/s41467-021-22330-w  

   Zhang, Yingxiao ; Ren, Qiurong ; Tang, Xu ; Liu, Shishi ; Malzahn, Aimee A. ; Zhou, Jianping ; Wang, Jiaheng ; Yin, Desuo ; Pan, Changtian ; Yuan, Mingzhu ; et al ( December 2021 , Nature Communications)

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   Abstract CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis . This study greatly expands the targeting scope of Cas12a for crop genome engineering. 

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2. Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants

   https://doi.org/10.1186/s13059-023-02929-6  

   Zhang, Liyang ; Li, Gen ; Zhang, Yingxiao ; Cheng, Yanhao ; Roberts, Nathaniel ; Glenn, Steve E. ; DeZwaan-McCabe, Diane ; Rube, H. Tomas ; Manthey, Jeff ; Coleman, Gary ; et al ( April 2023 , Genome Biology)

   Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.

   To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis inE. coliand identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency inT₀plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites.

   Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.

    

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3. Application of CRISPR/Cas9 to Tragopogon (Asteraceae), an evolutionary model for the study of polyploidy

   https://doi.org/10.1111/1755-0998.12935  

   Shan, Shengchen ; Mavrodiev, Evgeny V. ; Li, Riqing ; Zhang, Zhengzhi ; Hauser, Bernard A. ; Soltis, Pamela S. ; Soltis, Douglas E. ; Yang, Bing ( September 2018 , Molecular Ecology Resources)

   Tragopogon(Asteraceae) is an excellent natural system for studies of recent polyploidy. Development of an efficient CRISPR/Cas9‐based genome editing platform inTragopogonwill facilitate novel studies of the genetic consequences of polyploidy. Here, we report our initial results of developing CRISPR/Cas9 inTragopogon. We have established a feasible tissue culture and transformation protocol forTragopogon. Through protoplast transient assays, use of the TragCRISPR system (i.e. the CRISPR/Cas9 system adapted forTragopogon) was capable of introducing site‐specific mutations inTragopogonprotoplasts.Agrobacterium‐mediated transformation with Cas9‐sgRNA constructs targeting the phytoene desaturase gene (TraPDS) was implemented in this model polyploid system. Sequencing of PCR amplicons from the target regions indicated simultaneous mutations of two alleles and four alleles ofTraPDSin albino shoots fromTragopogon porrifolius(2x) andTragopogon mirus(4x), respectively. The average proportions of successfully transformed calli with the albino phenotype were 87% and 78% in the diploid and polyploid, respectively. This appears to be the first demonstration of CRISPR/Cas9‐based genome editing in any naturally formed neopolyploid system. Although a more efficient tissue culture system should be developed inTragopogon, application of a robust CRISPR/Cas9 system will permit unique studies of biased fractionation, the gene‐balance hypothesis and cytonuclear interactions in polyploids. In addition, the CRISPR/Cas9 platform enables investigations of those genes involved in phenotypic changes in polyploids and will also facilitate novel functional biology studies in Asteraceae. Our workflow provides a guide for applying CRISPR/Cas9 to other nongenetic model plant systems.

    

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4. Highly efficient CRISPR systems for loss-of-function and gain-of-function research in pear calli

   https://doi.org/10.1093/hr/uhac148  

   Ming, Meiling ; Long, Hongjun ; Ye, Zhicheng ; Pan, Changtian ; Chen, Jiali ; Tian, Rong ; Sun, Congrui ; Xue, Yongsong ; Zhang, Yingxiao ; Li, Jiaming ; et al ( June 2022 , Horticulture Research)

   Abstract CRISPR/Cas systems have been widely used for genome engineering in many plant species, while their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only few reports on application of CRISPR/Cas9 system in pear have been documented with a very low editing efficiency. Here, we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taken together, we build a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear. 

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5. Efficient and modular CRISPR‐Cas9 vector system for Physcomitrella patens

   https://doi.org/10.1002/pld3.168  

   Mallett, Darren R. ; Chang, Mingqin ; Cheng, Xiaohang ; Bezanilla, Magdalena ( September 2019 , Plant Direct)

   CRISPR‐Cas9 has been shown to be a valuable tool in recent years, allowing researchers to precisely edit the genome using an RNA‐guided nuclease to initiate double‐strand breaks. Until recently, classical RAD51‐mediated homologous recombination has been a powerful tool for gene targeting in the mossPhyscomitrella patens. However, CRISPR‐Cas9‐mediated genome editing inP. patenswas shown to be more efficient than traditional homologous recombination (Plant Biotechnology Journal, 15, 2017, 122). CRISPR‐Cas9 provides the opportunity to efficiently edit the genome at multiple loci as well as integrate sequences at precise locations in the genome using a simple transient transformation. To fully take advantage of CRISPR‐Cas9 genome editing inP. patens, here we describe the generation and use of a flexible and modular CRISPR‐Cas9 vector system. Without the need for gene synthesis, this vector system enables editing of up to 12 loci simultaneously. Using this system, we generated multiple lines that had null alleles at four distant loci. We also found that targeting multiple sites within a single locus can produce larger deletions, but the success of this depends on individual protospacers. To take advantage of homology‐directed repair, we developed modular vectors to rapidly generate DNA donor plasmids to efficiently introduce DNA sequences encoding for fluorescent proteins at the 5′ and 3′ ends of gene coding regions. With regard to homology‐directed repair experiments, we found that if the protospacer sequence remains on the DNA donor plasmid, then Cas9 cleaves the plasmid target as well as the genomic target. This can reduce the efficiency of introducing sequences into the genome. Furthermore, to ensure the generation of a null allele near the Cas9 cleavage site, we generated a homology plasmid harboring a “stop codon cassette” with downstream near‐effortless genotyping.

    

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